Neodihydrostreptomycin and acid addition salts



United States Patent 'NEODIHYDROSTREPTOMYCINFAND ACID ADDITION SALTS 'Teijiro Yabuta, Hiroshi; Ikeda, 'Kenji S hiroyanagi, Itsuo Fujimaki, and Mitsuhiko' Katayama, Tokyo, Japan NoDrawing. @APP icationApI'iLS, 1955, .Serial No. 500,306

' Claims priority, application Japan Marchfi, 1955 11 Claims. (Cl.-260--21,0)

This invention relates to Kthe preparation ofi-new" and therapeutically usefulantibioticsubstances, and particularly to the new antibiotic substance, neodihydrostrep- "tomycin, and acid salts thereof and the'processfor preparing the same by the reduction or acidsalts or streptomycin with amalgamated'aluminum.

'It is known that streptomycin is obtained fromelaboration products, 'formed during the "growth of "the .microorganism Actinomyces -griseus in a suitable culture medium, by adsorbing the "broth' filtrate -on carboxylic- --type cation exchange resin followed by elution witha mineral ==acid. ""The r product so 'obtained "is a strep- "tomycin salt, such *as a streptomycin trihydrochloride of relatively low potency or antibiotic activity. It can be further purified by solvent wmethod, fprecipitation --method and preferably "bycrystallization' method. in :copending application, Serial No. 306,738, filed August 27, l952,-we have disclosed that streptomycin interacts -with polyhalophenol to form -a salt-type combination -.(streptomycin-polyhalophenate) which is "sparingly soluble in water andvery'sbluble in certain organic solvents, and have i also disclosed that 'the streptomycinwpolyhalophenate may be 'Kdecompose'd by mineral-acids :rto recover the streptomycin -acid salts of high potency.

While the streptomycin acid -salts thus obtained are asintaform suitable for therapeutic use, it isfourid that .these i compounds t have certain inherent ff'disadvantages including .a rdegree of :chemical instability evidenced [particularly in: media containing *components capable of reacting wwith. carbonyl groups. Streptomycin contains .a carbonyl group demonstable with reagents such as .ssemicarbazide, .thiosemiearbazide, hydroxylamine and .rphenylhydrazine (Brink, Kuehl, Jn, =and Folkers, .eScience, 102, 506, 1945); and on' 'treatment with' alkali, it. is rapidlyfidegraded withloss "of its antibiotic activity (Barts, .Controu1is,-Crocks, Jr., and Rebstock, J. Am. Chem.,*Soc., .68, 2165, 1946),yielding streptidine (Ikeda, :Ikeda. .and Fujimaki, T I. Scientific *Research Inst.,Japan, :46, .255, 1952). Among-other -pro'ducts *formed on alkalinedegradation of streptomycin is maltol (Schenk wand "Spielman, I. Chem. Soc., 67, 2276, "1945); the formation of maltol being contingent onthepreswence-of a carbonyl group instreptomycin.

it is also known (Peck et-al,.,- J Chem. Soc.,"68, 11 390, 1946;' Bartz et "al., -ibid., 68,2163, 1946; Carboni and Regna, U. S. Patent 2,522,858, 1950; PeckfUfS. Patent 2,498,574, 1950) 'thabstreptomycin or acid addition salts .thereofis subjected tocatalytic hydrogena tion,one mole ofihydrogenper molegofstreptomycimis *taken up, and thatthe resulting 'dihydrostreptomycin: or acid addition salt thereof is antibioticallymctive .and is suitable for clinical application. Anelectrochemical procedure (Ohdakeet al., 'Repts. Sci. "Research Inst., Japan, 28, 103, 1952) and a sodium .borohydride method (Kaplan et al.,J. 'Am..Chem.TSOc., 76,5161, 1*954) forthe reduction of streptomycin have also been rreporte'd. Unlike -streptmycin, t'dihYdrostreptomycin :2 remains unchanged when treated 'with alkali z-atvroom temperature. Inasmuch as the productdoesnot react with carbonyl reagcnts'and is=-not .degraded byaa'lkali with the liberation .ofmaltol, it.-iis:clcar thatathetcar- 5 'bonyl group of streptomycin: hasFbeen reduced to car- "'binol.

An object of this invention is .tor provideatnew:' antibiotic: and the salte thereof. A further object is to rprepare a novel therapeutically active:saILofaneodihydrostreptomycin. i Further objects --willr appear;=hercinafter.

:It .is. now discovered,x according. to the present inven- .tion, .that when a :streptomycin saltvis treated with -amalgamated aluminum in man .:aqueous" or solvent xmedium atthe pH: range froms2z0 to:2z5, :the 'cor espending neodihydrostreptomycim (named byvus) zoom- -pond is formed.

wehave'also found that when. antacid. s'alt ofistrepto- :xl'llYCIl'll is: subjected to the cabove"mentioned:'reactionr at nthe pH. ranges of 8.-5 to 9.0an'd- 2.6 to .8..4,correspon ding. dihydrostreptomycinr salt and. the mixture of' n'eodi- ,:hydrostreptomycin :salt :and dihydrostreptomycin ris'alt are formed respectively.

'When dihydrostreptomycin is .heated in 'an 'aoid solution, its ultraviolet spectrogram showsradsorptionz'maxi- -mum-at-265 m (Hisoox,:Anal.w Chem., 23,923, 1951).

We have 1 found also that 'when 'neodihydrostrep'to- .mycin' is treatedasimilarly, no absorption is observed. r Hence, 'dihydrostreptomycin may be determined espectrophotometricallyas described above in the mixture of neodihydrostreptomycin and .dihydrostreptomycin.

The. results, given in-thei following Table :1 arei'prc- :sented to illustraterrthe. reaction 1 of our: invention.

TABLE 1 Dlhydro- 'Ne'odihydro- The pH of reaction mixture stre tomycin streptomycln o tamed obtained (percent) (percent) 0n the other hand," Fried and'Wintersteineri(U.T' S. Patent I2",552,547) have reported that dihydrostreptomycin may be obtained by subjecting.streptomycimto the action ofamalgamated aluminum in av non-alkaline (i. e., acid or neutral) medium. However, on the basis of the above mentioned our. discovery it. is evident that pure 'dihydrostreptomycinis not able to be obtained *un'derthe coridition=*-which Was descr'ibedby Fried--'and Wintersteiner.

The neodihydrostreptomycin-"compounds are approximately as active antibiotically as the streptomycin or dihydrostreptomycin= salts and are equally suitable; for clinicalapplication. 7 t

Neodihydrostreptomycin His .lchemically distinct :irom Tknown types .of .:streptomycin win .accordance with the followinglfacts and evidences. 5.111.contradistinctiomto 5 streptomycin .but r-similar .to Fdihydrostreptomycin, the

neodihydrostrostreptomycin .is --.not inactivated by .ex-

posure to alkali at room temperature, in rotherrwords, the degradation of the moleculeto. streptidine, .maltol and N-methylbglucosanaine, which ..is 1 brought about lby 'the action of this reagent ,on .streptomycin, L is 'fblocke'd as a consequence of the reduc'tivebhange; and "it *is clear that the carbonyl group of streptomycin (which resides in the maltol "gamma-pyrone forming moiety) has been reduced to carbinol.

Acid degradation of neodihydrostreptomycin produces streptidine and N-methyl-L-glucosamine components identical with thosegiven by streptomycin or dihydrostreptomycin; hence, the reduction with amalgamated aluminum involves only the streptose moiety. Furthermore, dihydrostreptomycin reacts with methanol containing dry hydrogen chloride to give in good yield the crystalline zit-methylpentaacetyl-dihydrostreptobiosaminide, M. P. 194 C. Neodihydrostreptomycin, under identical conditions, yields no crystalline compound.

Neodihydrostreptomycin is oxidized with sodium methaperiodate or periodic acid. Formaldehyde is formed and estimated by chromotropic acid (Vail and Bricker, Anal. Chem. 24, 975, 1952). Both of streptomycin and neodihydrostreptomycin give 0.6 mole of formaldehyde per mole; dihydrostreptomycin yields 1.6 mole.

Neodihydrostreptomycin can be resolved chromatographically from admixture with streptomycin and dihydrostreptomycin. For example, a paper strip may be spotted with a solution of the substances alone or in binary or ternary admixture. The strips are then developed chromatographically in accordance with known methods. The developed paper strips are then placed upon an agar plate, the surface of which has been inoculated with a test organism such as B. subtilis. After a period of time the positions of the resolved antibiotic substances are shown by separate zones of inhibition. The measured ratio of distance traveled along the strip for neodihydrostreptomycin compared with streptomycin was 0.3. The ratio of distance for neodihydrostreptomycin compared with dihydrostreptomycin was 0.7.

Other characteristic distinctions between neodihydrostreptomycin and dihydrostreptomycin are shown in Table 2.

TABLE 2 Dihydro- Neodihydrostreptomycin streptomycin M. P. (crystalline free base) 1 no M. P. up to 190-195", de-

300". tails are shown in Example 1. Potency (free base) units/mgm.:

biological 1,100. streptidine 3 920. {619" (free base; 0., 1% in water) 106.2

D50 in mgm. of base/kg. (mouse) 350. Nogueira's color reaction 4 negative. Ultraviolet spectrogram of the acid no absorption.

degraded substance. M. P. of the helianthate 225 215. M. P. of the reineckate 180. M. P. of the naphthalene-B-sulfonate. 173. M. P. of the p-(2-hydroxy-1-naphthyl- 224.

azo)-benzenesulfonate. a Methyl pentaacetyldihydrostrepneedle crystal no crystallizatoblosaminide. M. P. 194". tion.

1 All melting points were taken in the capillary and were uncorrected.

1 Rhodehamel et al., Science, 111, 233 (1950).

Sullivan and Hilmer, Amer. Chem. Soc. Abstracts of 109th Convention. Div. Biol. Chem, p. 413. 1946.

' Nogueira, Rev. port. farm, 1, 55 (1951): Chem. Abst., 9258 (1952).

Hiscox, Anal. Chem, 23, 923 (1951).

Regarded in certain of the broader aspects, novel features of the present invention comprise the new and therapeutically useful antibiotic substance of the class consisting of neodihydrostreptomycin and acid salts thereof .and the process for preparing the same by the reduction of acid salts of streptomycin with amalgamated aluminum (prepared according to Organic Synthesis, Collective Volume II, 397).

The acid salts contemplated in the present invention are acid salts in which the acid moiety resists reduction. Suitable acid salts are mineral acid salts and organic acid salts in which the acid moiety contains no reducible groups.

In carrying out the process in accordance with the present invention a quantity of an acid salt of streptomycin is dissolved in water, the pH is adjusted to 2.0-2.5 with dilute mineral or organic acid solution, in which is charged with a amalgamated aluminum and agitated for a time sufiicient to completely reduce the streptomycin to neodihydrostreptomycin (less than 1% residual streptomycin by maltol test) during which time the pH of the reaction mixture being adjusted to 2.0-2.5 with an acid solution. The amount of amalgamated aluminum used is not critical in the reaction although the rate of the reduction is increased or decreased with an increase or decrease in the amount of amalgamated aluminum. The amalgamated aluminum may be efiected in any medium which does not chemically affect streptomycin, inter alia, methanol, butanol or (preferably) water.

Various acids with which the pH of reaction mixture is adjusted are ordinary mineral acids and preferably those of which the ionization constants (K) are 10- 10.

As starting materials, it is possible to use impure concentrates of streptomycin salts, but it is preferred to use a substantially pure acid salt for the process.

After completion of the reduction, the aluminum compound is removed by filtration and the neodihydrostreptomycin may be obtained from the filtrate in the form of, or converted into, the free base or various acid addition salts thereof. The free base can be prepared from the above filtrate by subjecting it to a reaction with a base or basic hydroxide which forms substantially quantitatively the insoluble salts with the acid ions of the streptomycin salt and the acid with which the pH of the reaction mixture is adjusted. Thus, for example, reacting an aqueous solution of the mixture of neodihydrostreptomycin sulfate and sulfuric acid with the stoichiometric equivalent of barium hydroxide precipitates the sulfate ion as insoluble barium sulfate which is readily removed by filtration and the free base can then be recovered from the filtrate by crystallization from suitable aqueous-organic solvent (e.g. aqueous-acetone) or by freeze-drying. The various acid addition salts of neodihydrostreptomycin can be prepared from the above free base by neutralizing it with the acids. The product is, of course, obtainable in substantially-pure form directly by the reduction of substantially-pure streptomycin acid addition salt.

The acid addition salts of neodihydrostreptomycin can also be prepared directly from the above reaction filtrate by subjecting it (after neutralizing the free acid with an alkali which forms the soluble salt) to a reaction with an aqueous solution of an alkali salt of a polyhalophenol. The precipitated neodihydrostreptomycin-polyhalophenate is sparingly soluble in water. After washing the precipitate with water, the precipitate is dissolved in an waterimmiscible organic solvent and the neodihydrostreptomycin-polyhalophenate therein is converted into a water soluble salt of this antibiotic by intimately contacting the organic solvent solution with an aqueous water-soluble inorganic or organic acid, and recovering the aqueous phase, and freeze-drying it.

The following examples illustrate methods of carrying out the present invention, but it is to be understood that these examples are given by way of illustration and not of limitation.

Example I 46 gm. of streptomycin sulfate (790 units/mgm.) is dissolved in ml. of water. The pH is adjusted to 2.0 with dilute sulfuric acid and 10 gm. of amalgamated aluminum is added. The mixture is agitated at room temperature during which time the pH of the reaction mixture being adjusted to 2.0-2.5 with dilute sulfuric acid. After about two hours agitation, 2 gm. of amalgamated aluminum is added to increase the rate of reduction, the end-point of reduction being determined by assay for residual streptomycin by the maltol method (Boxer et.

c, 25.72; H, 4.06; N, 22.71.

.asoaeco :a1 JBiol. ..Chem., 169,5 3, 1947) After completion removed by "filtration. Tests for barium and sulfate ions lint Ethe .icleart filtrate .are ...negative. r'l he filtrate .is then mixed with three volumes of acetone causing slow separation of soft needle crystals weighing about 25 gm. The free base thus obtained, neodihydrostreptomycin, shows an activity of 1,100 units/mgm. (assayed biologically) and melts at l06-107 C. with a very small bubbling, resolidifying at about 110-120 C., and finally melts at about 190-195 C. with the formation of a brown-yellow color and with the evolution of a small amount of gas. The optical rotation is [ul 106.2 (C., 1% in water).

Analysis.--Calcd. for C21H41N'1O12: C, 43.28; H, 6.96; N, 16.81. Found: C, 42.97; H, 7.10; N, 16.35. Molecular weight: 583.6.

The neodihydrostreptomycin base obtained may also be recrystallized from hot water.

Example 11 Crystalline neodihydrostreptomycin base obtained as described in Example I, may be converted into various acid addition salts thereof.

(a) The sesquisulfate and trihydrochloride may be obtained by adding the equivalent amount of sulfuric acid or hydrochloric acid, respectively, to the base in aqueous solution, and freeze-drying the resulting solution.

(b) 1.2 gm. of neodihydrostreptomycin trihydrochloride obtained as described in the foregoing section, and having a potency of 890 units/mgm., is dissolved in 15 ml. of water and a solution of 2 gm. of ammonium reineckate in 110 ml. of water at 45 C. is added. On cooling neodihydrostreptomycin reineckate crystallizes in long needles melting with decomposition at 178-l80 C. (uncorrected) Analysis.-

Calcd. for C21H41N'zO1z.3HCr(SCN)4(NH3)2: Found: C, 25.77; H, 4.13; N, 23.08.

(c) 1.2 gm. of neodihydrostreptomycin trihydrochloride obtained as described in the foregoing section, and having a potency of 890 units/mgm., is dissolved in 15 ml. of water, and a hot solution of 1.8 gm. of methylorange in 70 ml. of 50% aqueous methanol is added. Orange crystals separate immediately from the solution and the product obtained after two recrystallizations from 50% aqueous methanol melted with decomposition at 215 C. (uncorrected).

Analysis.-Calcd. for C21H41N'10123(C14H15N303S)2 C, 50.46; H, 5.79; N, 14.94. Found: C, 50.67; H, 5.75; N, 14.91.

Example III 14 gm. of neodihydrostreptomycin sesquisulfate obtained as described in Example II is dissolved in 15 ml. of water. 4.5 gm. of sodium iodide, dissolved in 5 .ml. of water, is added and the solution is allowed to stand for several hours. The prismatic crystals of neodihydrostreptomycin-iodide-sulfate double salt are filtered and washed with cold water. The crystal is recrystallized from hot water.

Analysis.-Oalcd. for C21H41N1O12-HI-HSOJ, 15.99. Found: I, 15.85.

Example IV 80 gm. of partially purified streptomycinsulfate (700 units/mgm.) is dissolved in 500 ml. of water. The pH is adjusted to 2.0 with dilute phosphoric acid solution and 18 gm. of amalgamated aluminum is added. The mixture is then agitated mechanically at room temperature durhydroxide solution.

which 1 time. .the,..pI-I.of .thexeaction:.mixtureis.always adjusted: to..2.0-2:5.withdilutq phosphoric. acid. solution. After. twohours agitation, 3; gm. of. amalgamated alumi- .nuln..is: addedto increaseithe rateof reduction, .the end- ;pointof. reduction" being ..determined., by assay, for. resid- ..ual streptomycinby rthe...ma1t0l..method. After..compleltion. .of..the;. reduction, the..aluminum compound; is filtered .ol'if...and.lthe, pH. .is. adjusted .to 6L5. with...aqueous,.sodium 15% aqueous sodium pentachlorophenate solution is added to this filtrate under stirring until the test shows complete precipitation, the precipitate is Washed with water and separated by filtration. The precipitate is dissolved in ml. of n-butanol, insoluble impurities are filtered ofif and 10% of the aqueous sulfuric acid solution is added to the filtrate until the pH of the water layer is adjusted to 6.5. The water layer is recovered and poured into 300 ml. of methanol, the resulting precipitate of neodihydrostreptomycin sesquisulfate is recovered by filtration, washed with methanol and dried under reduced pressure. The product has an activity of 830 units/mgm.

Modifications maybe made in carrying out the present invention without departing from the spirit and scope thereof, and our invention is to be limited only by the appended claims.

We claim:

1. A substance of the class consisting of neodihydrostreptomycin and the acid addition salts thereof, said substance being produced by reacting an acid salt of streptomycin with amalgamated aluminum at a pH of 2.0 to 2.5.

2. Neodihydrostreptomycin produced by reacting an acid salt of streptomycin with amalgamated aluminum at a pH of 2.0 to 2.5.

3. Neodihydrostreptomycin sulfate produced by reacting an acid salt of streptomycin with amalgamated aluminum at a pH of 2.0 to 2.5.

4. Neodihydrostreptomycin hydrochloride produced by reacting an acid salt of streptomycin with amalgamated aluminum at a pH of 2.0 to 2.5.

5. Neodihydrostreptomycin iodide sulfate produced by reacting an acid salt of streptomycin with amalgamated aluminum at a pH of 2.0 to 2.5.

6. Neodihydrostreptomycin pentachlorophenate produced by reacting an acid salt of streptomycin with amalgamated aluminum at a pH of 2.0 to 2.5.

7. The process that comprises reacting an acid salt of streptomycin with amalgamated aluminum at a pH of 2.0 to 2.5, removing the salt component of neodihydrostreptomycin acid salt thus formed and the acid used for establishing the pH of the reaction mixture by adding stoichiometric equivalent of a base which forms an insoluble compound with said salt component and acid, filtering off the insoluble compound, and recovering from the filtrate the free base, neodihydrostreptomycin.

8. The process that comprises reacting streptomycin sulfate with amalgamated aluminum at a pH of 2.0 to 2.5, removing the sulfate component of the neodihydrostreptomycin sulfate thus formed and the sulfuric acid used to establish the pH of the reaction mixture by adding the stoichiometric equivalent of barium hydroxide and precipitating barium sulfate, filtering off the precipitate, and recovering from the filtrate the free base, neodihydrostreptomycin.

9. The process defined in claim 10, further comprising the step of neutralizing the neodihydrostreptomycin base with an acid solution and recovering the acid addition salt thereof.

10. The process of obtaining highly purified neodihydrostreptomycin salt which comprises reacting a partially purified acid salt of streptomycin with amalgamated aluminum at the pH range from 2.0 to 2.5 and treating the reaction filtrate with an alkali salt of a polyhalophenol to form a salt-type combination product of neodihydrostreptomycin and the polyhalophenol, recovering 7 5 said product, and converting the product into a waterrecovering the product, and converting it into the neodisoluble acid addition salt of the neodihydrostreptomycin. hydrostreptomycin sulfate.

11. The process of obtaining highly purified neodihydrostreptomycin sulfate which comprises reacting par- References Cited in the tile of this patent tially purified streptomycin sulfate with amalgamated 5 UNITED STATES PATENTS aluminum at the pH range from 2.0 to 2.5 and treating the reaction filtrate with sodium salt of pentachlorophenol to form the neodihydrostreptomycin-pentachlorophenate,

2,552,547 Fried et a1. May 15, 19571 

1. A SUBSTANCE OF THE CLASS CONSISTING OF NEODIHYDROSTREPTOMYCIN AND THE ACID ADDITION SALTS THEREOF, SAID SUBSTANCE BEING PRODUCED BY REACTING AN ACID SALT OF STREPTOMYCIN WITH AMALGAMATED ALUMINUM AT A PH OF 2.0 TO 2.5. 